Mouthwash use and oral cancer: a systematic review and meta-analysis

ABSTRACT OBJECTIVE This study aimed to investigate the effect of mouthwash use on the development of oral cancer. METHODS Observational studies with adult/older adult populations that have examined the association between mouthwash use and oral cancer were included. Electronic search was performed in July 2022, with no time or language restrictions. PubMed/Medline, Embase, and Web of Science databases were used, and the search was extended to theses and dissertations libraries, Google Scholar, reference lists, and other sources. Methodological quality was assessed using the Newcastle-Ottawa Scale and quantitative data synthesis was performed by random effects meta-analysis, with different subgroup analyses and meta-regression. This revision was registered in Prospero (CRD42020143307). RESULTS Of the 4,094 studies identified in the search, 15 case-control studies were included in the review, totaling 6,515 cases and 17,037 controls. The meta-analysis included 17 measures of effect from 15 case-control studies. The pooled OR was 1.00 (95%CI: 0.79–1.26, n = 17 studies), but it was 2.58 (95%CI: 1.38–4.82, n = 2 studies) among those who had used mouthwashes three times or more times a day, and 1.30 (95%CI: 1.10–1.54, n = 4 studies) among those who had used mouthwashes for more than 40 years. CONCLUSIONS We found evidence that a high frequency of mouthwash use may be associated with an increased risk of oral cancer. However, despite the biological plausibility for this association, we suggest caution upon interpretation of our findings due to the few number of studies that have investigated the mouthwash use frequency, which should be considered. Therefore, we recommend that future studies assess, in detail, the frequency, duration, and content of mouthwashes to increase the strength of evidence for a possible dose-response effect of mouthwashes on oral cancer risk.


INTRODUCTION
Oral cancer (OC) comprises tumors of the lip, oral cavity, and oropharynx 1 .It is considered a major public health problem worldwide 2 , being responsible for 476,125 new cases in 2020 3 .Squamous cell carcinoma represents more than 90% of this total 4 , commonly affecting men after the fifth decade of life 5 .OC is a complex and multifactorial etiology disease 5 , in which cells accumulate oncogenic stimuli and deviation from homeostatic mechanisms.Thus, a transition process from a normal to a dysplastic epithelium can be triggered by potentially malignant precursor disorders for the carcinoma 6 .Some of the major risk factors are tobacco use 7,8 , alcohol consumption 1,9 , age 10 , and sex 11 , as well as oral human papillomavirus infection, diet, genetics 12 , and persistent exposure to pathological or environmental cytotoxics 13 , without consensus about the mouthwashes use.
Mouthwashes have been used for centuries as breath fresheners, medicines, and antiseptics 14 but the safety of their use and a likely association with OC have been widely discussed [15][16][17][18][19][20][21] .Different hypotheses have been investigated for the mechanisms involved in the carcinogenicity of alcohol-based mouthwashes, such as (1) intraoral oxidation of ethanol to its toxic metabolite acetaldehyde 17,22 , and (2) an accentuated local cytotoxic effect on human epithelial keratinocytes of the oral mucosa 13,23 .Cytotoxicity occurs when ethanol, in contact with the cells, induces deeper-layers stem cells to divide more often than normal to replace the damaged epithelium, leading to a variety of cancer-related errors, thereby increasing the risk of malignant transformation 23 .
The preponderant role of ethanol in the carcinogenic potential of alcoholic mouthwashes does not exclude the possibility that other components may also be involved in OC 13 .The impact of the complex mixture on oral cell's cytotoxicity and antimicrobial activity is largely unknown 24 .Various molecules included in commercial mouthwashes are preparations created and proposed for the market 25 .In this way, it is possible that active antibacterial ingredients, other than ethanol, such as phenolic compounds 26 , triclosan 27,28 , cetylpyridinium chloride 29 , and chlorhexidine [30][31][32] may increase the risk of OC by changing the diversity of oral bacteria 15 and causing cell damage 24 .
A previous systematic review 33 and meta-analyses [34][35][36] have investigated the association between mouthwash use and OC, but none of them found any evidence.The authors did not perform subgroup analyses considering adjusted and unadjusted estimates, type of controls, or frequency and duration of mouthwash use.Only Houstiuc et al. 34 performed analyses in terms of duration and frequency of mouthwash use and alcohol content, but they only considered upper aerodigestive tract cancers, not OC.
In addition, although the searches have included the grey literature and reference lists, they were restricted to the main online databases, especially PubMed/ Medline, Web of Science, and Scopus.The PICO, PECO, or PEO strategies were not mentioned and few descriptors were inserted, and only studies published in English 34 or English and Spanish 35 were included.Furthermore, some of these meta-analyses 34,35 included studies that may have contained overlapping samples [37][38][39][40][41][42][43] .This potential duplication occurred because these studies were part of multicenter research 44,45 or were smaller in scale [37][38][39][40][41][42][43] .Moreover, the meta-analyses incorporated various types of studies, such as case series 46 , metaanalysis 36 , and studies focused on outcomes or objectives unrelated to oral cancer 17,[47][48][49][50][51][52][53][54] .Therefore, since some studies indicate an association between mouthwash use and OC 15,44,45,[55][56][57] , whereas other studies do not show such association, and considering the gaps left behind by previous meta-analyses, we propose to estimate the pooled effect of mouthwash use on OC depending on duration and frequency, type of control, and adjustment for confounding factors.
Initially two examiners were responsible for the search (JSSA, EBAFT).The PEO search strategy [Population (adults or older adults), Exposure (mouthwash use), and Outcome (OR)] was used.Thus, objective-related keywords, and MeSH terms (Medical Subject Headings) combined with Boolean operators (OR/AND/NOT) were used to ensure that the search strategy was comprehensive.The titles were searched in July 2022.Year of publication and language were unrestricted.The search strategy by database is detailed in Supplementary Table 1 a .The searched study titles and their respective information were included in a Microsoft Excel ® 365 software spreadsheet (Microsoft Corporation, Washington, USA) to check for duplicity and to apply the eligibility criteria.Duplicate studies were excluded.The searches were compared, and any disagreement was resolved by the third reviewer (MCFNC).

Eligibility Criteria
We included primary studies with adult or older adult populations that aimed to analyze the association between mouthwashes and OC.The excluded criteria included: 1) studies with specific populations with syndromes or congenital changes; 2) studies with more susceptible populations to the development cancer such as those previously exposed to chemotherapy or radiotherapy, and patients with specific genetic mutations; 3) publications involving the same population sample -in this case, the study with the a Available from: https://drive.google.com/file/d/1XhrSZK83w25gs21xPjPXbBngWiBQ8aXf/ view?usp=sharing https://doi.org/10.11606/s1518-8787.2023057004752major sample was selected; 4) studies with outcomes defined as dysplasia, cell damage, or nuclear alterations; and 5) letters to the editor, conference and congress abstracts, case series, case reports, in vitro studies, experimental studies in animals, review studies, and meta-analyses.

Selection of Studies
An independent selection of studies was performed by two examiners (JSSA, EBAFT) and disagreements were resolved by consensus with the third reviewer (MCFNC).The first selection was based on the title and abstract, hiding the journal and author's names, avoiding possible bias and conflicts of interest.Studies not selected at this stage or in the subsequent stages were registered in the spreadsheet as excluded, with their respective reasons.In cases where the study seemed to be eligible, but presented insufficient data in the title and abstract, the text was fully read and evaluated following the inclusion criteria afterwards.The full texts of the remaining studies were recovered and those eligible for this review were identified.

Data Extraction
Relevant data from the selected articles were extracted, processed, and tabulated in a data collection form pre-developed in Microsoft Excel ® 365 (Microsoft Corporation, Washington, USA) by two reviewers (JSSA, EBAFT).All included articles were case-control studies, and the following data were recorded: authors of the studies, year of publication, country, recruitment period, sample size, age, gender (only one or two genders), type of exposure (mouthwash use -yes or no -and according to the frequency of use, alcohol content, and use duration over the years), type of outcome (OC site and ICD), type of controls (community or hospital), effect size (odds ratio), case-controls ratio, and variables considered in the adjustment for confounding (whether in pairing, sample restriction, or adjusted analysis).
For studies that reported measures of effect according to the cancer involvement site (oral cavity, pharynx, larynx, esophagus), those located in the oral cavity or oral cavity and pharynx were selected (when the measure of effect was simultaneously presented at both sites).For studies that reported effect sizes by categories regarding frequency of use or time of use, these measures were considered in subgroup analysis for dose response, sometimes being recategorized to allow comparability with other studies.For results stratified by gender, the measures of effect from each stratum were considered in the meta-analysis by inserting the letters a (men) and b (women).For studies that presented adjusted estimates for different confounding variable arrangements, the effect size adjusted for the largest number of variables was considered instead of potential mediators.Considering the possibility of residual confounding, subgroup analysis was performed considering three categories of adjustment: adjusted, when adjusted, at least, for age, gender, and tobacco and alcohol consumption; partially adjusted, when adjusted only for some of these variables; or unadjusted.Alcohol content could not be categorized, as information was missing in some studies, possibly because it was selfreported data.Data missing from the studies were disregarded.When the study did not present enough data to be included in the quantitative analysis, e-mails were sent to the authors to retrieve the data.

Risk of Bias and Grading Quality of Evidence
The individual risk of bias of each study included in the systematic review was assessed by the Newcastle Ottawa Scale (NOS) for case-control studies by two independent examiners (JSSA, EISM).Differences were resolved by consensus in the presence of the third reviewer (MCFNC).The quality of evidence of the studies included in the meta-analysis was assessed following the GRADEpro Guideline Development Tool (GDT) 62,63 .

Statistical Analysis
Stata 14.0 software (StataCorp, College Station, USA) was used for the meta-analysis.Since the heterogeneity evaluated by the I 2 test was high (77.1%), the DerSimonian-Laird Random-Effect method was chosen.Subgroup analyses were done with the studies that reported duration and frequency of mouthwash use to assess a likely dose-response, as well as to evaluate the subgroup according to the type of control (hospital or community) and the variables considered in the confounding adjustment.
Crude and multivariable meta-regressions were used to assess the contribution (%) of the co-variables [gender (men only; women only; and men and women), setting (low/middle-income or high-income country), sample size (up to 500; 501 to 1,000; and over 1,000 subjects), cancer site (only oral cavity or oral/pharyngeal/larynx sites), control type (hospital or community), and case-controls ratio (at least one case to two controls "1:2" or one case to one control "1:1"), OR adjustment] on the heterogeneity among the studies.Co-variables with p-value < 0.20 in crude meta-regression were included in the multivariable meta-regression.A funnel plot associated with the Egger regression asymmetry test was used to investigate the possibility of publication bias.OR were estimated and weighted by the study sample size and by their respective 95% confidence intervals (95%CI).

Searching Results
We identified 4,094 records in the bibliographic search.After excluding duplicates, 3,517 titles and abstracts were read.Of these, 50 studies were selected for full-text reading, and 14 studies were included in our review, with one more paper identified after searching in the reference lists.Thus, 15 papers were included in the qualitative and quantitative analyses, totalizing 6,515 cases and 17,037 controls.The reasons for the 36 full-text articles excluded were: sample already included in other multicenter studies 44,45 (n = 7); letter to editor (n = 4); insufficient data (n = 2); other outcome/ objective (n = 12); in vitro studies (n = 3); review (n = 5); conference abstract (n = 3) (Figure 1).
Except for Sharma et al. 55 , Mashberg et al. 58 , and Young et al. 59 , the other studies were matched minimally by gender and age.Other prevalent confounding variables included in multivariable analyses comprised tobacco and alcohol consumption, and, less often, fruit and vegetable consumption, ethnicity, socioeconomic conditions, among others.Human papillomavirus (HPV) was not included in the regression analyses of the identified studies.One study restricted the sample to people aged 40 years or older with no history of tobacco use 65 .One study provided only crude effects, i.e., no matching, no restriction, and no multivariable analyses 55 .

Risk of Bias and Grading Quality of Evidence
According to NOS, eight studies presented a low risk 15,44,45,57,64,67,70,71 , and seven presented a moderate risk of bias 55,56,65,66,68,69,72  weakness of the investigated studies is their control selection since they present hospital controls.In addition, none of the studies reported the blinding of cases and controls in regarding exposure, which could have generated measurement bias.
The quality of evidence in the studies included in this meta-analysis, according to GRADEpro GDT, was low (Supplementary Figure 1 c ).The low quality of the evidence was especially due to the inclusion of observational studies (case-control), in which there is a higher risk of bias due to the impossibility of randomizing the exposure, and because of the inconsistency present in the studies.

Meta-Analysis
Figure 2A shows the summarization of the 17 OR from the 15 studies included in the meta-analysis.Mouthwash use, regardless of alcohol content or frequency/duration of use, was not associated with OC (OR = 1.00; 95%CI: 0.79-1.26)and the heterogeneity among studies was substantial (I 2 : 77.1%).The funnel plot suggests a possible effect of the smaller studies, as they are more concentrated on the bottom right, but the Egger tests were not statistically significant (p = 0.651), indicating symmetry in the distribution of studies,   and therefore a low possibility of publication bias (Figure 2B).When considering only the five effect estimates (OR) of the studies that analyzed alcohol-containing mouthwash versus no mouthwash use (Figure 2C), the overall weighed random effect increased but remained non-significant (OR = 1.20; 95%CI: 0.93-1.55).

Meta-Regression
In the non-adjusted analysis, the co-variable 'setting' and 'case-control ratio' presented a p < 0.20 in association with OC, and explained 23.8% and 26.3% of the heterogeneity among  the studies, respectively.Multivariable meta-regression showed that these variables, together, explained 39.4% of heterogeneity among the studies (Supplementary Table 3 d ).

DISCUSSION
In this systematic review and meta-analysis of 15 case-controls and 17 OR estimates including 6,515 cases and 17,037 controls, we observed no association between mouthwash use (any versus no use) and OC (OR = 1.00; 95%CI: 0.79-1.26).Three previous meta-analyses also did not find association [34][35][36] .When Hostiuc et al. 34   difference in risk between cases and controls was not significant.Argemi et al. 37 also did not find association between mouthwash use and OC, neither when considered mouthwashes with alcohol in five case-control studies, nor without alcohol in four studies.Similarly, Gandini et al. 36 estimated a non-significant relative risk summarized from nine studies.These authors also considered any frequency/duration of mouthwash use.
However, when we investigated the frequency of use, the odds of developing OC in individuals who frequently used mouthwashes (three or more times a day) was 1.30 times higher than in those who never used (OR = 1.30; 95%CI: 1.10-1.54);additionally, it was 158% higher among those who used mouthwashes for more than 40 years when compared to non-users (OR = 2.58; 95%CI: 1. 38-4.82).This could suggest a dose-response effect.Gandini et al. 36 , however, estimated the relative risk with the frequency of use once, twice, or thrice a day and found no significant trend in risk with increasing daily use.Comparably, Hostiuc et al. 34 found a non-statistically significant risk difference on the incidence of cancers in upper aerodigestive tract according to the frequency of use.We were not able to identify other meta-analyses that had assessed the dose-response effect related to OC. Tobacco, alcohol, and betel consumption, diet, nutrition, as well as immunosuppression, environmental, and genetic factors are considered risk factors for OC 61,73 .When we performed subgroup analyses considering studies that reported both crude and adjusted associations, we reduced the probability that confounding biased the pooled estimates.However, the possibility of unmeasured confounding cannot be completely disregarded since important confounding factors could have been disregarded, such as HPV infection (not considered in any of the studies), tobacco and alcohol consumption, diet/nutrition, and socioeconomic conditions (considered only in some of the association estimates).Additionally, if a confounding factor is poorly measured or inadequately defined, residual confounding may also occur.However, we can suppose that the effect of the time of mouthwash use could be confounded by the age of the participants since increasing age is associated with increasing OC risk 10 .However, all studies included in this subgroup analysis have been adjusted for age and other potential confounders 45,67,69 .
Over the years, the main hypothesis for the link between mouthwashes and OC was the alcohol composition of these products.The carcinogenesis process would occur inducing a marked cytotoxic effect in human epithelial keratinocytes 13,23 , previously investigated in vitro with two commercially available mouthwash brands 14 .For each brand, an alcoholfree and an alcohol-containing version (96 mg/mL and 213.03 mg/mL, respectively) were tested on human oral keratinocytes with and without a mild dysplasia.The authors concluded that alcohol-based mouthwashes were genotoxic to both normal and dysplastic oral keratinocytes, inducing generalized changes in gene expression in vitro.
Similar results were also found in clinical trials evaluating the effect of alcohol-containing and alcohol-free mouthwashes on exfoliated oral cells 74,75 .In this context, the authors found an increased frequency of micronuclei and cellular abnormalities in the group exposed to the alcohol-containing mouthwash.Due to the superficial and intracellular characteristics of the oral mucosa epithelium, the detection of DNA damage and cell death in desquamated epithelial cells requires the genotoxic agent to overcome the permeability barrier of the basal layer and induce DNA damage, later converting them into micronuclei during cell division 76 .The correlation between the number of stem cell divisions that occurred in a tissue during a person's life and the risk of cancer diagnosis in that tissue is highly positive and statistically significant 77 .
When considering only the use of alcohol containing mouthwashes versus no use, the association in our meta-analysis did not remain significant (OR = 1.20; 95%CI: 0.93-1.55).Argemí et al. 35 also summarized data referred to the alcohol content of nine studies and showed a non-significant association (OR = 1.48; 95%CI: 0.85-2.56).Although the composition of mouthwashes and the alcohol content were not well described in all studies, the supposition that these non-alcoholic products with antimicrobial activity may also be cytotoxic should be mentioned 25,78 .A wide variety of antiseptics containing different active ingredients are available and widely used in dentistry 30 .These products are regulated as cosmetic products, thereby not requiring ingredients declaration 26 .Thus, we can assume that other components are also involved in cell damage 24 or oral microflora alterations, harboring the potential to alter the balance of immune tolerance, further contributing to the genesis and promotion of OC 15 .
The most common molecules contained in mouthwashes are chlorhexidine, essential oils, cetylpyridinium chloride, triclosan, octenidine, delmopinol, polyvinylpyrrolidone, hyaluronic acid, and natural compunds 25 .When exposed to human gingival fibroblasts at the concentration required to inhibit 50% of cellular metabolic activity (IC50), 0.2% chlorhexidine decreased the viable cells number and increased the number of cells undergoing apopstosis 30 .Other in vitro studies 78-81 corroborated these findings.Cetylpyridinium chloride was also found to exhibit severe cytotoxic effects against human keratinocytes and murine fibroblasts even at low concentrations 29 .Listerine ® , a product that contains thymol, eucalyptus, methyl salicylate, and menthol, had its cytotoxicity evaluated 26 and the authors have suggested all phenolic compounds may contribute, to some extent, to cell damage in vitro.
Triclosan is toxic to mitochondria, immune cells 27 , and possibly to the neural system 28 .In 2017, the Colgate-Palmolive company removed triclosan from dentifrices, following a determination by the United States Food and Drug Administration 82 .In addition to triclosan, twenty-three other active ingredients have also been removed from over-the-counter antiseptic products, due to insufficient data on their safety and effectiveness.
Hereupon, a limitation of our meta-analysis was the failure to perform subgroup analyses according to the different proportions of mouthwashes alcohol content.Otherwise, we could assess whether the substances present in their formulations are important for OC regardless of the alcohol content since the available evidence is supported only by in vitro studies.Thus, new studies that present data regarding the alcohol content of mouthwashes and their main components are essential to investigate and clarify the impact these molecules have.This meta-analysis was also the first to analyze the quality of evidence using the GRADEpro GDT 62,63 .The tool estimated the quality of evidence as low.This result is mainly due to the design of the included studies.Case-control is the most feasible type of study design to investigate this subject, but it presents more biases than clinical trials and cohort studies.In this context, the possibility of some confounding, measurement, and selection biases leads us to classify the risk of bias as 'serious' by GRADEpro, despite most studies being classified as moderate or low risk of bias according to the NOS criteria.However, due to the unfeasibility of randomization, we can admit certain risk of bias in the case-control studies, so we can suggest that the NOS instrument, adopted in this meta-analysis, could have underestimated the risk of bias in the included studies.However, NOS is one of the most used instruments 83 , and its content validity and interobserver reliability are well established 83,84 .A recent meta-analysis on the topic 34 did also use the same instrument; moreover, NOS seems to provide the same reliability, varying in applicability, compared to the ROBINS-I tool recommended by Cochrane.Furthermore, the complexity of using the ROBINS-I tool can be a limiting factor for its adoption 83 .Another factor that decreases the quality of the evidence is the inconsistency of results since some studies have showed positive (risk) 45,67 and others negative (protective) 55,56 associations between mouthwash use and OC.
As strengths of our study, this meta-analysis was the first to consider the effect of the frequency and duration of mouthwash use over the years in OC.Despite pioneer, our findings should be carefully interpreted, given the small number of studies that considered the frequency (n = 2) and duration (n = 3) of mouthwash use.Another strength was the vast bibliographic search in a higher number of databases, including the grey literature, https://doi.org/10.11606/s1518-8787.2023057004752 using the PEO strategy, without language and publication date restrictions.Therefore, we were able to reach studies that were not included in the previously published metaanalyses.In addition, we did not include, in this meta-analysis [37][38][39][40][41][42][43] , samples previously used in other larger studies 44,45 .We considered the alcohol content of mouthwashes versus the non-use when conducting the analyses, and different subgroup analyses were also performed.Lastly, a meta-regression was performed to explain the heterogeneity.

CONCLUSIONS
This systematic review and meta-analysis showed no relationship between mouthwash use and OC, except for the mouthwash use for three or more times a day and for people who have used it for over 40 years, suggesting a possible dose-dependent effect.These findings, however, should be analyzed with caution given the small number of studies that consider the frequency of mouthwash use.Therefore, we recommend that future studies evaluate, in detail, the frequency, duration, and content of mouthwashes to increase the strength of evidence for a possible dose-response effect of this exposure on OC risk.

Figure 1 .
Figure 1.Study selection process evaluating the use of mouthwash and oral cancer.
(B) The funnel plot for this meta-analysis.The orange line represents the adjusted line corresponding to the Egger asymmetry regression test.(C) Meta-analysis of random effects of oral cancer odds ratio.a Gender male.b Gender female.

Figure 3 .
Figure 3. Meta-analysis of random effects of oral cancer odds ratio among mouthwash users and non-users, subgroup analysis according to the effect measure adjustment (A), control type (B) and usage time (C).

Figure 4 .B
Figure 4. Meta-analysis of random effects of oral cancer odds ratio among mouthwash users and nonusers considering the frequency of use < 1 time a day (A), 1 to 2 time a day (B) 3 or more time a day (C).
Characteristics of the studies included in the systematic review and meta-analysis of the mouthwash use and oral cancer.
evaluated the overall risk of upper aerodigestive tract cancers associated with mouthwash use in 17 studies, the authors reported that the